Combinations of immunosupressive agents for the treatment or prevention of graft rejections

ABSTRACT

A pharmaceutical composition useful in the treatment or prevention of transgenic xenograft rejection comprising immunosuppressant compounds selected from the group consisting of an IL-2 transcription inhibitor and immunosuppressant compounds that immunosuppress for B-cell-mediated or antibody-mediated rejection of xenografts, and pharmaceutically acceptable diluents or carriers, and a method of preventing hyperacute rejection, reducing early graft damage, improving early xenograft function and promoting long term survival of said transgenic xenografts comprising the steps of i) contacting the body fluid removed from a human recipient with a xenoantigenic material which is bound to a biocompatible solid support, ii) reintroducing the treated body fluid into the recipient, and iii) treating the recipient with said pharmaceutical composition.

This is a continuation of International Application No. PCT/EP00/04250,filed May 10, 2000, the contents of which are incorporated herein byreference.

This invention is concerned with certain novel pharmaceuticalcompositions comprising combinations of immunosuppressive agents, theuse of such compositions for the treatment or prevention of xenograftrejection, and novel therapies for facilitating transplantation ofxenogenic tissues or organs into humans and to promote long termsurvival of said tissues or organs.

In order for xenotransplantation to present a clinically viabletreatment for organ disease, it is necessary to effectively treat orprevent acute rejection and chronic rejection of donor organs. Effectivetreatments need to inhibit T-cells, and also B-cell-mediated orantibody-mediated rejection.

However, a problem associated with combining compounds that suppressT-cells as well as compounds that act against B-cell-mediated rejectionor antibody-mediated rejection is the potential and unpredictablepharmacokinetic interaction of the compounds which may influence thetoxicity or the immunosuppression of the combination.

Surprisingly, the applicant has found that pharmaceutical compositionscomprising a certain combination of immunosuppressant compounds displaygood tolerability whilst at the same time display prolonged xenograftsurvival of donor organs, few rejection episodes and good graftfunction.

The invention provides in one aspect a method of treatment or preventionof xenograft rejection which comprises administering of at least twoimmunosuppressant compounds indepentally selected from the groupconsisting of (a) IL-2 transcription inhibitors and (b)immunosuppressant compounds that immunosuppress for B-cell-mediated orantibody-mediated rejection of xenografts.

Pharmaceutical compositions useful in the treatment or prevention ofxenograft rejection comprise at least two immunosuppressant compoundsselected from the group consisting of (a) IL-2 transcription inhibitorsand (b) immunosuppressant compounds that immuno-suppress forB-cell-mediated or antibody-mediated rejection of xenografts.

Therefore, the invention provides in another aspect a pharmaceuticalcomposition useful in the prevention or treatment of xenograft rejectioncomprising combinations of immuno-suppressant compounds selected fromthe group consisting of (a) IL-2 transcription inhibitors and (b)immunosuppressant compounds that immunosuppress for B-cell-mediated orantibody-mediated rejection of xenografts.

In another aspect, the group of immunosuppressant compounds consists of(a) an IL-2 transcription inhibitor and (b) immunosuppressant compoundsthat immunosuppress for B-cell-mediated or antibody-mediated rejectionof xenografts.

As used herein, including the claims, the combinations ofimmunosuppressant agents cover the administration of the agents forsimultaneous, separate or sequential use. Thus e.g. they may be in apackage or in a blister.

The term “IL-2 transcription inhibitor” refers to immunosuppressivecompounds whose immunosuppressive activity derives principally or insignificant part from their direct or indirect inhibition of IL-2 genetranscription, e.g. corticosteroids, ascomycins and cyclosporines, FK506and their various derivatives and analogues.

Cyclosporine, (also known as cyclosporin A or cyclosporin) is animmunosuppressive cyclic undecapeptide. Its structure is disclosed, e.g.in the Merck Index, 11th edition; Merck & Co. Inc., Rahway, N.J., USA(1989) under listing 2759. Formulations of cyclosporine are commerciallyavailable under the trademark SANDIMMUN or SANDIMMUNE and amicroemulsion preconcentrate formulation of cyclosporine is sold underthe trademark NEORAL or OPTORAL.

Immunosuppressant compounds that immunosuppress for B-cell-mediated orantibody-mediated rejection of xenografts include rapamycin and/orderivatives thereof including 40-O-(2-hydroxyethyl)-rapamycin, ormyriocin analogues such as2-amino-2-[2-(4-octylphenyl)-ethyl]-1,3-propanediol, or mycophenolicacid (MPA) or pharmaceutically acceptable salts thereof, orcyclophosphamide.

A preferred rapamycin derivative is 40-O-(2-hydroxyethyl)-rapamycin.40-O-(2-hydroxyethyl)-rapamycin is a rapamycin derivative the structureof which is disclosed in WO 94/09010, example 8, and is a semi-syntheticderivative of rapamycin. The structure of rapamycin is given inKesseler, H., et al.; 1993; Helv. Chim. Acta; 76; 117, and numerousimmunosuppressive derivatives and analogues of rapamycin are known.

MPA sodium salt is known and is disclosed in published patentapplication No. WO 97/38689. Even more preferred is a MPA sodium salt inform of a formulation as described in U.S. Pat. No. 6,025,391 which isincorporated herein by reference.

More preferred pharmaceutical compositions according to the inventioncomprise combinations of a pharmaceutically acceptable salt ofmycophenolic acid (MPA), for example the sodium salt of MPA, rapamycinand/or derivatives thereof including 40-O-(2-hydroxy-ethyl)-rapamycin,and IL-2 transcription inhibitors.

Most preferred pharmaceutical compositions according to the inventioncomprise double combinations of MPA sodium salt and cyclosporine or40-O-(2-hydroxyethyl)-rapamycin, the double combination of cyclosporineand 40-O-(2-hydroxyethyl)-rapamycin, or a triple combination of MPAsodium salt, cyclosporine and 40-O-(2-hydroxyethyl)-rapamycin.

Pharmaceutical compositions according to the invention actsynergistically, i.e. the immuno-suppressive effect of the combinationof compounds is greater than additive. This has the advantage thatrelatively low doses of each compound may be used in the pharmaceuticalcompositions. Synergy may be calculated according to a method describedin Berenbaum, Clin. Exp. Immunol. (1977) 28:1.

The indications for which the pharmaceutical compositions are useful areconditions associated with, or causal to, transplant rejection, forexample treatment (including amelioration, reduction, elimination orcure of etiology or symptoms) or prevention (including substantial orcomplete restriction, prophylaxis or avoidance) of xenograft rejection,including acute and chronic rejection of an organ when the organ donoris of a different species from the recipient, most especially rejectionmediated by B-cells or antibody-mediated rejection.

Preferably, in one group of embodiments there is no IL-2 transcriptioninhibitor present. In another group of embodiments, one IL-2transcription inhibitor is present, and yet in another group ofembodiments, two or more IL-2 transcription inhibitors are present.

More particularly the method comprises the administration of apharmaceutical composition comprising a combination of apharmaceutically acceptable salt of MPA, for example the sodium salt ofMPA, rapamycin and/or derivatives thereof including40-O-(2-hydroxyethyl)-rapamycin, and IL-2 transcription inhibitors.

Preferably the method comprises the administration of a pharmaceuticalcomposition comprising a combination of MPA sodium salt and one or moreimmunosuppressant compounds selected from the group consisting of (a) anIL-2 transcription inhibitor, especially cyclosporine, and (b) rapamycinand/or derivatives thereof, especially 40-O-(2-hydroxyethyl)-rapamycin.

In a further aspect of the invention there is provided the use of apharmaceutical composition comprising compounds selected from the groupconsisting of (a) an IL-2 transcription inhibitor and (b)immunosuppressant compounds that immunosuppress for B-cell-mediated orantibody-mediated rejection of xenografts, in the treatment of acondition as hereinabove described.

More particularly the invention provides the use of a pharmaceuticalcomposition comprising a combination of a pharmaceutically acceptablesalt of MPA, for example the sodium salt of MPA, rapamycin and/orderivatives thereof including 40-O-(2-hydroxyethyl)-rapamycin, and IL-2transcription inhibitors, in the treatment of a condition as hereinabovedescribed.

Preferably the invention provides the use of pharmaceutical compositionscomprising combinations of MPA sodium salt and one or moreimmunosuppressant compounds selected from the group consisting of (a) anIL-2 transcription inhibitor, especially cyclo-sporine, and (b)rapamycin and/or derivatives thereof, especially40-O-(2-hydroxyethyl)-rapamycin, in the treatment of a condition ashereinabove described.

In yet another aspect of the invention there is provided a kit-of-partscomprising any of the pharmaceutical compositions hereinabove described,especially a pharmaceutical composition comprising MPA sodium salt andone or more immunosuppressant compounds selected from the groupconsisting of (a) an IL-2 transcription inhibitor, especiallycyclosporine, and (b) rapamycin and/or derivatives thereof, especially40-O-(2-hydroxyethyl)-rapamycin, together with instructions for use inthe treatment or prevention of a condition as hereinabove described.

The dosages of the compounds will vary depending on the individual to betreated, the route of administration and the nature and severity of thecondition to be treated. For example, in the prevention or treatment ofxenograft rejection, an initial dose of about 2 to 3 times themaintenance dose may suitably be administered about 4 to 12 hours beforetransplantation, followed by a daily dosage of 2 to 3 times themaintenance dosage for one to two weeks, before gradually tapering downthe dose at a rate of about 5% per week to reach the maintenance dose.

The exact dosage of each compound may be determined having regard to theparticular therapeutic blood levels required for each compound. Thus,through judicious selection of the dosage of each compound, surprisinglyit has been found that pharmaceutical compositions may be formed thatare tolerated and which display synergistic action in immunesuppression.

Accordingly, in another aspect of the invention there is provided apharmaceutical composition comprising (a) an IL-2 transcriptioninhibitor, in particular cyclosporine, that may be delivered to apatient at a dosage such that the 16 hour blood trough level is e.g. upto 1500 ng per ml, e.g. 50 to 1500, e.g. 50 to 1000 ng per ml, e.g. upto 500 ng per ml, , e.g. 50 to 500 ng per ml, more particularly 100 to500 ng per ml, e.g. 300 to 500 ng per ml, and (b) an immunosuppressantcompound that immunosuppresses for B-cell-mediated or antibody-mediatedrejection of xenografts that may be delivered to a patient at a dosagesuch that the 16 hour blood trough level is e.g. 0.1 to 20, e.g. 0.1 to10, e.g. 1 to 10, preferably 3 to 6 μg per ml of the active substance,or is e.g. 1 to 90, e.g. 5 to 50, e.g. 10 to 35 ng per ml, moreparticularly 10 to 20 ng per ml.

In particularly preferred pharmaceutical compositions, apharmaceutically acceptable salt of MPA, for example the sodium salt ofMPA, may be delivered to a patient at a dosage such that the 16 hourblood trough level is e.g. 0.1 to 20, e.g. 0.1 to 10, e.g. 1 to 10,preferably 3 to 6 μg per ml of MPA. IL-2 transcription inhibitors, e.g.cyclosporine may be delivered to a patient at a dosage such that the 16hour blood trough level of, e.g. cyclosporine is e.g. up to 1500 ng perml, e.g. 50 to 1500, e.g. 50 to 1000 ng per ml, e.g. up to 500 ng perml, for example 50 to 500 ng per ml, more particularly 100 to 500 ng perml, e.g. 300 to 500 ng per ml. Rapamycin and/or derivatives thereof,e.g. 40-O-(2-hydroxyethyl)-rapamycin may be delivered to a patient at adosage such that the 16 hour blood trough level of, e.g.40-O-(2-hydroxyethyl)-rapamycin is e.g. 1 to 90, e.g. 5 to 50, e.g. 10to 35 ng per ml, more particularly 10 to 20 ng per ml.

The blood concentrations hereinabove described may be determinedaccording to any convenient method known in the art. For example, bloodmay be collected in EDTA-coated containers, and detection of bloodlevels may be carried out by, e.g. radioimmunoassay or by ELISA.Detection of MPA is suitably carried out after protein precipitationusing acetonitrile using an HPLC method with UV detection at 305 nm.From the data collected in this way, the blood trough levels may becalculated by methods known in the art.

Having regard to the blood trough levels stated hereinabove, the skilledperson may determine those dosages that provide a therapeutic amount ofcompound at a level that is tolerated and which exhibits synergisticaction in immune suppression.

The weight ratio of component compounds of the pharmaceuticalcompositions may vary having regard to the desired blood trough levelsstated hereinabove.

Pharmaceutical compositions may comprise combinations of (a) an IL-2transcription inhibitor and (b) an immunosuppressant compound orcompounds that immunosuppress for B-cell-mediated or antibody-mediatedrejection of xenografts in a weight ratio of e.g. about 1:50 to 1000:1,e.g. about 1:50 to 500:1, e.g. about 1:50 to 200:1, more particularly1:2 to 50:1, e.g. 10:1. When more than one compound thatimmunosuppresses for B-cell-mediated or antibody-mediated rejection ofxenografts is employed, for example in the case of a triple combination,the combined weight of said immunosuppressant compounds is reflected inthe aforementioned weight ratio.

In a preferred embodiment a pharmaceutical composition comprises MPAsodium salt and cyclosporine in a weight ratio of e.g. about 1:0.03 toabout 1:2, e.g. about 1:0.03 to about 1:0.5.

In another preferred embodiment a pharmaceutical composition comprisesMPA sodium salt and 40-O-(2-hydroxyethyl)-rapamycin in a weight ratio ofabout 1:0.0005 to 0.015, to 1:0.001 to 0.0075, in particular, 1:0.0025.

In a particularly preferred pharmaceutical composition comprising thedouble combination of MPA sodium salt and cyclosporine, MPA sodium saltmay be applied at a dosage of e.g. 10 to 200, e.g. 10 to 100 mg/kg/day,preferably 20 to 60 mg/kg/day, in particular 40 to 60 mg/kg/day; whereascyclosporine may be applied at a dosage of e.g. 10 to 100, e.g. 10 to 50mg/kg/day, preferably 10 to 15 mg/kg/day, in particular 3 to 6mg/kg/day. Most preferably MPA sodium salt may be applied at a dosage of20 mg/kg/day and cyclosporine may be applied at a dosage of 10mg/kg/day.

In another particularly preferred pharmaceutical composition comprisinga double combination of MPA sodium salt and40-O-(2-hydroxyethyl)-rapamycin, MPA may be applied at dosages referredto in the preceding paragraph, whereas 40-O-(2- hydroxyethyl)-rapamycinmay be applied at a dosage of from 0.05 to 1.5 mg/kg/day, e.g. 0.1 to0.75 mg/kg/day, e.g. 0.25 to 0.5 mg/kg/day. Most preferably MPA sodiumsalt may be applied at a dosage of 20 mg/kg/day and40-O-(2-hydroxyethyl)-rapamycin may be applied at a dosage of 1.5mg/kg/day.

In yet another particularly preferred pharmaceutical compositioncomprising a double combination of cyclosporine and40-O-(2-hydroxyethyl)-rapamycin, cyclosporine may be applied at a dosageof e.g. 10 to 100, e.g. 10 to 50 mg/kg/day, preferably 10 to 15mg/kg/day, in particular 3 to 6 mg/kg/day; whereas40-O-(2-hydroxyethyl)-rapamycin may be applied at a dosage of 0.05 to1.5 mg/kg/day, e.g. 0.1 to 0.75 mg/kg/day, e.g. 0.25 to 0.5 mg/kg/day.

In yet another particularly preferred pharmaceutical compositioncomprising the triple combination aforementioned, MPA sodium salt may beapplied at a dosage of e.g. 10 to 200, e.g. 10 to 100 mg/kg/day,preferably 20 to 60 mg/kg/day, in particular 40 to 60 mg/kg/day;cyclosporine may be applied at a dosage of e.g. 10 to 100, e.g. 10 to 50mg/kg/day, preferably 10 to 15 mg/kg/day, in particular 3 to 6mg/kg/day. 40-O-(2-hydroxyethyl)-rapamycin may be applied at a dosage offrom 0.05 to 1.5 mg/kg/day, e.g. 0.1 to 0.75 mg/kg/day, e.g. 0.25 to 0.5mg/kg/day. Most preferably MPA sodium salt may be applied at a dosage of20 mg/kg/day, and 40-O-(2-hydroxyethyl)-rapamycin may be applied at adosage of 1.5 mg/kg/day. Cyclosporine may be applied at a dosage of 10mg/kg/day.

The dosages referred to hereinabove may be administered to a patient inany convenient way, for example individual dosages referred tohereinabove may be administered daily in 2 divided doses. Any regimenmay be used, provided that therapeutic amounts of the individualcompounds are delivered to the patient.

In larger mammals, for example humans, an indicated daily dosage for MPAsodium salt is in the range of 0.5 to 2.0 g/day, e.g. about 1.5 g/day,cyclosporine is in the range of from about 25 mg to about 1000 mg perday, preferably 50 mg to 500 mg per day, and40-O-(2-hydroxyethyl)-rapamycin is in the range from about 0.25 mg toabout 15 mg per day.

The application of the pharmaceutical composition may be preceded by theadministration of a suitable induction therapy, chosen from any suitableinduction therapy known in the art, for example a short course ofcyclophosphamide, e.g. up to 40 mg/kg, e.g. 20 to 40 mg/kg i.v. per dayfor 4 days. Furthermore, a tapering course of steroids, e.g.methylprednisolone at a concentration of 1 mg/kg at day one tapering toa baseline of 0.2 mg/kg/day may be administered.

The compounds hereinabove described may be used in pharmaceuticalcompositions according to the invention in free or fixed combination,preferably in free combination. By ‘free’ is meant that each compound isformulated separately in a discrete dosage form. By ‘fixed’ is meantthat the compounds are formulated together in one carrier. As a furtherembodiment, the pharmaceutical compositions may be both free and fixedwhereby two or more compounds may be formulated in a single carrierwhereas a further compound of the pharmaceutical composition may beformulated as a discrete dosage form.

Where one or more of the compounds are formulated separately, theindividual dosage forms may be taken together or substantially at thesame time (e.g. within fifteen minutes or less) so that, in the case oforal administration for example, said compounds are presentsimultaneously in the stomach.

The pharmaceutical composition according to the invention may beformulated in any convenient dosage form, the component compounds beingeither in a single carrier or formulated as discrete dosage forms as ina free combination, for example oral dosage forms, e.g. solid oraldosage forms or solutions or dispersions, or in forms suitable forintravenous administration.

Pharmaceutical compositions for oral administration of, e.g.cyclosporine and/or 40-O-(2-hydroxyethyl)-rapamycin, are suitablyemulsions, microemulsions, preconcentrates of either, or soliddispersions, especially water-in-oil microemulsion preconcentrates oroil-in-water microemulsions.

Cyclosporine may be formulated in any of the ways known in the art, inparticular the known microemulsion preconcentrate formulations ofcyclosporine are particularly suitable for use in the present invention.

40-O-(2-hydroxyethyl)-rapamycin may be formulated in any of the waysknown in the art, for example as a microemulsion (see WO 96/13273), as afat emulsion for use in intravenous administration (see WO 97/25977), asa suspension (see WO 96/13239) or as a solid oral dosage form, forexample, as a co-precipitate with a suitable carrier medium (a so-calledsolid dispersion) as more fully described in WO 97/03654 all of whichdocuments are incorporated herein by reference.

MPA sodium salt may be formulated in any of the ways described in WO97/38689 which is incorporated herein by reference, in particular as asolid oral dosage form, e.g. an enteric-coated tablet.

Pharmaceutical compositions according to the invention are useful astherapies in the treatment or prevention of xenograft rejection,including acute or chronic rejection of an organ from a discordantspecies, e.g. heart, lung, combined heart-lung, liver, kidney, isletcells, most especially when the rejection is mediated by B-cells orantibodies.

Whereas the pharmaceutical compositions are useful in the treatments asset forth in the preceding paragraph, nevertheless a significantobstacle in the successful treatment of patients with theafore-mentioned pharmaceutical compositions is the prevention oramelioration of hyperacute rejection.

Hyperacute rejection is the first immunological barrier to thetransplantation of a donor organ from discordant species into humans. Itoccurs when the recipient's own immune system attacks and destroys thetransplanted organ, usually within minutes or within a number of hours.Hyperacute rejection occurs in a xenograft because humans havepre-formed xenoreactive antibodies which bind to the animaltissue,activating the human complement cascade and leading to graftdamage. Accordingly, organ transplants from animal species, such as apig, into humans may not be viable unless treatments are available thatprevent hyperacute rejection.

Means for ameliorating hyperacute rejection are known in the art. Forexample, organs from pigs transgenic for human decay-accelerating factor(hDAF) may not be hyperacutely rejected upon transplantation intonon-human primates. Similarly, extracorporeal removal of xenoantibodiesfrom a recipient's blood is also known in the treatment of hyperacuterejection, see for example U.S. Pat. No. 5,817,528 or U.S. Pat. No.5,651,968.

However, whereas hyperacute rejection may be prevented in this manner,antibodies may nevertheless trigger damage to the donor organ andthereby compromise the organ leading to poor early graft function orpremature organ failure notwithstanding the administration ofimmunosuppressive therapies.

The applicant has now found that long term survival of a transgenicdonor organ may be enhanced if a recipient receives treatment to removexenoantibodies extracorporeally as well as receiving appropriateimmunosuppressive drug therapy.

Accordingly, the invention provides in another aspect a method oftreating a patient in need of such therapy comprising i) exposing thebody fluid removed from a human recipient with a xenoantigenic materialor anti human mono- or polyclonal antibodies or an other antibodyadsorbent, which is bound to a biocompatible solid support, ii)reintroducing the treated body fluid into the recipient, and iii)treating the recipient with immunosuppressive drug therapy comprising acombination of immunosuppressant compounds selected from the groupconsisting of (a) IL-2 transcription inhibitor and (b) immunosuppressantcompounds that immunosuppress for B-cell-mediated or antibody-mediatedrejection of xenografts.

In another aspect, the group of immunosuppressant compounds consists of(a) an IL-2 transcription inhibitor and (b) immunosuppressant compoundsthat immunosuppress for B-cell-mediated or antibody-mediated rejectionof xenografts.

Methods and materials for carrying out the steps i) and ii) referred toabove are known in the art and suitable examples are described in U.S.Pat. No. 5,817,528 or U.S. Pat. No. 5,651,968, or publishedinternational application PCT EP98/02227 each of which are incorporatedherein by reference.

In a particularly preferred aspect of the invention, the step i) may becarried out using an Ig-Therasorb® column thereby selectivelyimmunoadsorbing IgM, IgG and IgA.

The step i) is usually carried out pre-operatively. However,alternatively or additionally it may be used perioperatively orpost-operatively in parallel with the immunosuppressive drug therapy.Such post-operative use may be used if during treatment a build up ofxenospecific antibodies is detected. Judicious use of the step i) andii) pre-operatively, perioperatively and/or post-operatively in parallelwith the immunosuppressive drug therapy may contribute significantly tothe long term survival of donor organs.

In addition to the improvement of the early graft function, effectivetreatments need to inhibit T-cells and also B-cell-mediated orantibody-mediated rejection. Accordingly, preferred immunosuppressivedrug therapies for use in step iii) comprise any of the pharmaceuticalcompositions, dosages and other aspects as hereinabove described.

There now follows a series of examples which are illustrative of theinvention.

EXAMPLE 1 Organ Xenograft Studies: Porcine Kidney to Cynomolgus Monkey

Cynomolgus monkeys undergo heterotopic renal transplants using porcinekidneys transgenic for hDAF.

Immunosuppression consists of induction therapy consisting ofcyclophosphamide, Neoral® and a tapering course of steroids followed bymaintenance therapy with a free combination of cyclosporine and MPAsodium salt as set forth in Table 1.

Induction therapy consists of four non-consecutive daily intravenousinjections of cyclo-phosphamide (40 mg/kg), Neoral® andmethylprednisolone low dose treatment (1 mg/kg) day one and thereafterreducing the dose each day by 0.05 mg/kg, and subsequently to a baselinedose of 0.02 mg/kg/day as part of the maintenance therapy.

Maintenance therapy, subsequent to the induction therapy, consists ofcyclosporine. (Neoral®) and MPA sodium salt (MPA Na in Table 1) in theform of a powder in a 1% methylcellulose (Courtauld's Chemicals)solution.

Dosing occurs twice daily at 8 am and 4 pm and the doses set forth inTable 1 are equally divided for that purpose. Dosing is carried out bygastric gavage under slight ketamine anaesthesia (10 mg/kg) in a volumeof 2 ml/kg of body weight followed by flushing with at least 10 ml/kgphysiological saline. TABLE 1 Tolerability of a combination of MPASodium and Cyclosporine in Cynomolgus Monkeys. (Dosages in mg/kg). MPANa Cyclosporine² Outcome  50 bid 25 bid Tolerated 100 bid 25 bidTolerated 40 + 60¹ 30 bid Tolerated¹First dose at 7 am, second at 3 pm²Neoral ®bid = Twice per day

Significant prolongation of the xenografts treated with the foregoingcombinations were observed compared with monotherapy of the individualcompounds of the combinations.

EXAMPLE 2 Organ Xenograft Studies: Hamster-to-Rat

Donor male Chinese hamsters are obtained from Tongji Medical University.

Recipient male SD rats are obtained from Tongji Medical University.

Maintenance therapy consists of MPA sodium in the form of a powder in a1% methylcellulose (Courtauld's Chemicals) solution, cyclosporine(Neoral®), and/or 40-O-(2-hydroxyethyl)-rapamycin in the form of a soliddispersion at 9.09% by weight, together with HPMC (81.82% by weight) andlactose (9.09% by weight).

All compounds are freshly prepared before administration and dissolvedin distilled water. The compounds are administered daily per oral bygavage (<2 ml/kg body weight).

Anaesthesia is carried out using Hypnorm and Hypnovel i.v. anaesthesia.TABLE 2 Table 2 illustrates significant prolongation of xenografts usingcompositions according to the invention. Treatment Schedule (mg/kg/day)Graft survival (days) n MPA Sodium salt (20) 5, 5, 6, 6, 6, 14 6Cyclosporine A (10) MPA Sodium salt (20) 6, 7, 7, 11, 16, 17 640-O-(2-hydroxyethyl)-rapamycin (1.5) MPA Sodium salt (20) 6, 7, 10, 10,12 5 40-O-(2-hydroxyethyl)-rapamycin (1.5) Cyclosporine A (10)n: number of patients

EXAMPLE 3 Use of Immunoadsorntion in Heterotopic Xenotransplantation ofhDAF Pig Hearts to Baboons

Baboons undergo heterotopic heart transplants using porcine heartstransgenic for hDAF.

Materials for Immunosuppression are:-

Cyclosporin A (CyA): (Sandimmun®) given via intramuscular injection at aconcentration of 100 mg/ml; and Optoral® given by oral gavage at 100mg/ml.

Cyclophosphamide (CyP): Endoxan® for injection at 200 mg/ml

Mycophenolate sodium: In a form describe in Example 2 above.

Methylprednisolon (MPS): Urbason® in a 40 mg vial.

Prednisolon (PDN): Prednesol® as a 5 mg tablet.

Post-operative anesthesia is carried out using buprenorphinehydrochloride. Nausea and vomiting are treated using metoclopramide.

Animals are dosed twice a day with a 12 hour interval. Animals receivethe following regimen: CyA is applied initially as i.m. injection at adose of 25 mg/kg/day after surgery. On the first post-operative day 20mg/kg is applied i.m. In the afternoon Optoral is given by oral gavageat a dose of 100 mg/kg. Thereafter doses are modified according to CyAtrough levels aiming at >1500 ng/ml.

CyP is given i.v. on the day before surgery at 40 mg/kg, on the day ofsurgery at 20 mg/kg and on the second post-operative day at 20-30 mg/kg.An additional dose may be given up to 20 mg/kg on day 4. The last dosemay be modified according to WBC and platelet count. Thereafter, CyP isadministered only for rejection treatment.

Mycophenolic acid sodium is given orally twice a day to ensure troughlevels of 3-6 μg/ml.

MPS is given at the time of surgery at a dose of 1 mg/kg i.v. On thefollowing two days the same dose will be applied orally and thereafterthe dose is reduced to 0.05 mg/kg/day until a baseline of 0.2 mg/kg isreached.

Body weight of the animals is taken during morning dosing . Food isprovided one hour after morning dosing and water is freely available.

For Xenotransplantation of transgenic organs immunoadsorption is carriedout pre-operatively using an Ig-Therasorb® column. According to thexenoreactive natural antibody titre, between 6 and 14 cycles are carriedout withdrawing blood from a central venous catheter.

The treatment was well tolerated and the xenografts exhibited good longterm survival.

1. A pharmaceutical composition useful in the treatment or prevention ofxenograft rejection comprising (a) cyclosporin; (b) an immunosuppressantcompound selected from the group consisting of mycophenolic acid, apharmaceutically acceptable salt of mycophenolic acid, and combinationsthereof; and (c) rapamycin and/or derivatives thereof.
 2. Apharmaceutical composition comprising (a) cyclosporin; (b) animmunosuppressant compound selected from the group consisting ofmycophenolic acid, a pharmaceutically acceptable salt of mycophenolicacid, and combinations thereof; and (c) rapamycin and/or derivativesthereof as a combined preparation for simultaneous, separate orsequential use in the treatment or prevention of xenograft rejection. 3.A kit of parts comprising a pharmaceutical composition according toclaim 1 together with instructions for use in the treatment orprevention of xenograft rejection.
 4. Use of a pharmaceuticalcomposition according to claim 1 or 2 in the manufacture of a medicamentfor the treatment or prevention of xenograft rejection.
 5. Apharmaceutical composition according to claim 1 or 2 wherein thepharmaceutically acceptable salt of mycophenolic acid is MPA sodium saltformulated as an enteric-coated solid oral dosage form.
 6. A method forthe treatment or prevention of xenograft rejection comprisingadministering a pharmaceutical composition according to claim 1 or
 2. 7.A method for reducing early graft damage, improving early xenograftfunction or promoting long term survival of xenografts of transgenicorgan in human recipients comprising: (i) exposing body fluid removedfrom a human with a xenoantigenic material or anti-human mono- orpolyclonal antibodies or another antibody adsorbent, which is bound to abiocompatible solid support; (ii) reintroducing the treated body fluidinto the human, and (iii) treating the human with a compositioncomprising at least two immunosuppressant compounds selected from thegroup consisting of (a) IL-2 transcription inhibitors and (b)immunosuppressant compounds that immunosuppress for B-cell-mediated orantibody-mediated rejection of xenografts.
 8. A method for reducingearly graft damage, improving early xenograft function or promoting longterm survival of xenografts of an organ transgenic for hDAF in humanrecipients comprising: (i) exposing body fluid removed from a human witha xenoantigenic material or anti-human mono- or polyclonal antibodies oranother antibody adsorbent, which is bound to a biocompatible solidsupport; (ii) reintroducing the treated body fluid into the human, and(iii) treating the human with a composition comprising at least twoimmunosuppressant compounds selected from the group consisting of (a)IL-2 transcription inhibitors and (b) immunosuppressant compounds thatimmunosuppress for B-cell-mediated or antibody-mediated rejection ofxenografts.
 9. The method according to claim 7 wherein the steps (i) and(ii) are conducted postoperatively and in parallel with treatment withthe composition.